Fibroblast Activator, Collagen Production Promoter, Collagen Contraction Promoter, Hyaluronic Acid Production Promoter, ATP Production Promoter, Melanin Formation Inhibitor, and Agent for External Application to the Skin
a technology of collagen production and activation, which is applied in the direction of biocide, drug composition, peptide/protein ingredients, etc., can solve the problems of reducing the elasticity and flexibility of the skin and the like, sagging, and not essentially improving the aging skin,
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example 1
[0047]The present inventors performed a fibroblast activating action evaluation test by the MTT reduction method and using normal skin fibroblasts derived from human (see TIM Mosmann; Journal of Immunological Methods p 55-63, 1983).
[0048]Using DMEM (Gibco) containing 5% FBS (fetal bovine serum; purchased from NICHIREI CORPORATION), normal skin fibroblasts derived from human (manufactured by KURABO INDUSTRIES LTD.) were plated on a 96 well plate at a density of 2×104 cells / well, and cultured at 37° C., 5% CO2 for 24 hr. After removing the medium, the cells were washed with PBS(−) (NISSUI PHARMACEUTICAL CO., LTD.), the medium was changed to DMEM containing 1% FBS supplemented with each concentration of conagenin, and the cells were cultured at 37° C., 5% CO2. The blank was MEM containing 1% FBS, which was free of test specimen. After culture for 48 hr, the absorbance at 550 nm was measured by MTT reduction method, based on which the MTT reduction amount was determined. The results are...
example 2
Type I Collagen Production Promoting Effect of Conagenin on Human Skin Fibroblasts
[0050]Normal human skin fibroblasts (manufactured by KURABO INDUSTRIES LTD.) were added to a 24 well plate at 5×104 cells per well. Using Medium 106S medium (manufactured by KURABO INDUSTRIES LTD.) containing 2% FBS, the cells were cultured under an atmosphere of 95% (V / V) air-5% (V / V) carbon dioxide gas at 37° C. for 24 hr. The medium was changed to Medium 106S medium (free of FBS) containing each sample, and the cells were cultured under the same conditions for 24 hr. After the completion of culture, the culture supernatant was taken to evaluate the type I collagen biosynthesizability and, to count the cells, the cells were harvested by a trypsin treatment. The samples used were two kinds of conagenin contents of 0.1 μM, 1 μm, 10 μM, 100 μM, 1 mM (final concentration), and magnesium L-ascorbyl phosphate (manufactured by Wako Pure Chemical Industries, Ltd., final concentration 100 μM) as a positive co...
example 3
Collagen Contraction Promoting Effect of Conagenin for Human Skin Fibroblasts
[0052]A collagen solution (collagen was used trade name I-AC manufactured by KOUKEN Co., Ltd.) dissolved in normal human skin fibroblasts (manufactured by KURABO INDUSTRIES LTD., 1×105 cells / ml) was prepared on ice according to the explanation insert attached to the product, and the collagen was gelled in 6 wells at 37° C. Thereafter, 0.25% FBS / DMEM medium containing a sample (PBS as conagenin or negative control) was added. The gel was detached from the dish wall, and the level of contraction of the gelled collagen was examined. As a positive control, DMEM medium containing 10% FBS instead of 0.25% FBS was used. The medium was changed every 2 days and, one week later, the medium was removed by suction. The diameter of the collagen gel was measured, the area was calculated, and the area ratio was compared to that of the negative control as 1. The results are shown in Table 3.
TABLE 3collagen gel area ratione...
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